Recombinant beta-galactosidase derived from Streptococcus pneumaniae

ABSTRACT

The present invention relates to a novel beta-galactosidase derived from  Streptococcus pneumoniae , a BgaC protein exhibiting the enzyme activity, and a method for using the same. The protein can be used in the modification and analysis of sugar chain and used as an anti-cancer agent.

This application claims priority to Korean Patent Application Ser. No.KR 10-2006-0057140, filed Jun. 23, 2006, which is incorporated herein byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to an enzyme, BgaC protein havingbeta-galactosidase (EC 3.2.1.23) activity derived from Streptococcuspneumoniae, and a method for using the same.

2. Description of the Related Art

Beta-galactosidase is a cleavage enzyme that belongs to the family 35 ofglycohydrolases, and that is found in plants and animals, as well as ina wide variety of microorganisms such as yeasts, fungi, bacteria, andarchaea. Beta-galactosidase hydrolyzes lactose and its structurallyrelated compounds, and additionally catalyzes transgalactosylationreaction of various beta-D-galactopyranosides including lactose. Thehydrolase and transferase activity of beta-galactosidase are useful forindustrial applications (Nkayama and Amachi, 1999; Hung and Lee, 2002).Beta-galactosidase is widely used in the hydrolysis of lactose, which ispresent in milk products such as milk and whey, to glucose andgalactose. The hydrolysis process is a fundamental method for reducingthe lactose content employed in the food and dairy industries (Greenbergand Mahoney, Process Biochem. 16: 2-8, 1981; Gekas and Lopez-levia, 20:2-12, 1985).

Meanwhile, many studies have been made on the relationship between thestructure and function of a sugar chain in order to investigate itsbiological meaning and role, which has attracted a great deal ofinterest in the field. For the studies, there is a need for analyzingeach sugar chain at the level of linkage specificity. In general, forthe analysis of a component or sequence of a sugar chain, equipment suchas HPLC and mass spectrometry has been widely used. However, alinkage-specific glycosidase is essentially needed forlinkage-specifically analyzing the structure of a sugar chain.Therefore, there is a trial that employs a beta-galactosidase havinglinkage-specific glycosidase activity for analyzing the structure of asugar chain.

Further, with respect to cancer treatment, there are a variety oftreatment methods, such as chemotherapy administrating variousanti-cancer agents, immunotherapy promoting antibody production againstcancer cells, surgical therapy removing cancer cells, and radiationtherapy killing cancer cells by irradiating radioactive rays. However,even though the primary cancer has been eradicated, some problems maystill exist. That is, cancer may be called as a malignant tumor becauseof its metastatic ability, and in many cases, metastatic cancer is morelikely to cause death. It cannot be said yet that a method forinhibiting the metastasis of cancer cells has been established, and amedicine having the effects of inhibiting the metastasis of cancer cellshas not yet been commercially available. On the other hand, severalsteps are considered for the metastasis mechanism, and a casualrelationship between the cancer metastasis and the sugar chain has beenrecently discussed in academic meetings. In the metastasis of cancercells, cancer cells are first released from a cancer-developed site, andthen move through the blood stream in a human body. E-selectin, which isone of intercellular adhesive molecules, is expressed on the surface ofan intravascular endothelial cell for several reasons. This E-selectininteracts with free cancer cells moving through the blood stream in thehuman body, and causes a rolling phenomenon, in which the free cancercells roll on the surfaces of the intravascular endothelial cells andreduce their moving speed in the blood. Consequently, the free cancercells adhere to the intravascular endothelial cells, and then passthrough the intravascular endothelial cells. Thus, the cancer cellsenter the vascular tissue, resulting in a new cancer cell nest formed.

In the series of steps, the adhesion between the E-selectin, which isone of the intercellular adhesive molecules expressed on the surface ofthe intravascular endothelial cell, and the sugar chains, which arepresent on the surfaces of cancer cells, plays a very important role inan initial stage of the adhesion between the cancer cells and theintravascular endothelial cells. As a sugar chain antigen on the surfaceof cancer cells, which interacts with the E-selectin, a sialicacid-containing complex sugar chain, called as Sialyl Lewis X (sLe^(x))and Sialyl Lewis A (sLe^(a)), has been identified. That is, there is areport that the sugar chain acts as a ligand involved in the metastasisof cancer cells (Takada et al., Cancer Res. 53: 354-361, 1993). In thesugar chains, galactose-beta1,3-N-acetylglucosamine (Gal-β1,3-GlcNAc) isa core polysaccharide of sLe^(a), andgalactose-beta1,3-N-acetylglucosamine andgalactose-beta1,3-N-acetylgalactosamine (Gal-β1,3-GalNAc) are majorcomponents of mucin-type and complex-type glycoproteins. Accordingly, aspecific galactosidase capable of cleaving the terminal galactose atbeta1,3 linkage is needed for inhibiting metastasis.

It has been known that BgaC protein has an activity of cleavingnon-reducing terminal galactose linked by beta1,3 glycosidic linkage.However, BgaC proteins, which are galactosidases capable of cleaving thegalactose linked by beta1,3-linkage, have not been well known, untilnow. Among the proteins, there is no enzyme that specifically recognizesa sugar followed by a galactose to cleave the galactose.

On the other hand, when an infectious microorganism invades a host cell,its glycosidase is involved in the cleavage of sugar chain exposed onthe surface of the host cell. Therefore, the related genes are oftenfound in pathogenic microorganisms. There is a report that agalactosidase having the activity of cleaving beta 1,4 linkage, which isdesignated as BgaA, was found in Streptococcus pneumoniae causingpneumonia. The Streptococcus pneumoniae BgaA is a putative 2,235-aminoacid protein having a molecular weight of 247.3 kDa, which is present onthe cell surface, and purified from culture medium (Zahner andHakenberck, J. Bacteriol. 182: 5919-5921, 2000; Glasgow et al., J. Biol.Chem. 252: 8615-8623, 1977; Hughes and Jeanloz, Biochemistry, 10:1535-1548, 1964).

The present inventors have analyzed the genomic information ofStreptococcus pneumoniae that has already been disclosed (Hoskins etal., J. Bacteriol. 183: 5709-5712, 2001). As a result, it was found thatanother new putative nucleic acid molecule having galactosidase activityexists, thereby performing functional analysis of the nucleic acidmolecule. Consequently, a novel cleavage enzyme, which has differentsugar chain specificity from BgaA, was detected.

In particular, it was found that the novel enzyme selectively recognizesonly the sugar chain having galactose-beta1,3-N-acetylglucosaminelinkage and hydrolyzes the galactose, which is useful for the functionalanalysis and modification of a sugar chain.

Further, it was observed that treatment of the enzyme inhibits thecolony formation of cancer cells, which offers a possibility of usingthe enzyme as an anti-cancer agent, thereby completing the presentinvention.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a protein exhibitingbeta1,3-galactosidase activity, which has an amino acid sequencerepresented by SEQ ID NO: 2 or an amino acid sequence having at least90% homology therewith.

It is another object of the present invention to provide an isolatednucleic acid molecule encoding the protein.

It is still another object of the present invention to provide arecombinant vector containing the nucleic acid molecule.

It is still another object of the present invention to provide atransformant capable of expressing the beta1, 3-galactosidase byintroducing the recombinant vector.

It is still another object of the present invention to provide a methodfor selectively cleaving a non-reducing terminal galactose ofgalactose-beta1,3-N-acetylglucosamine using the beta1,3-galactosidase.

It is still another object of the present invention to provide a methodfor analyzing the structure of a sugar chain using the method forlinkage-specifically cleaving the sugar chain.

It is still another object of the present invention to provide a methodfor treating or diagnosing a disease, in which the sugar chainspecifically occurred in the disease such as cancer is cleaved using themethod for linkage-specifically cleaving the sugar chain.

It is still another object of the present invention to provide a methodfor inhibiting cancer cell growth using the protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a drawing showing PCR amplification of a bgaC gene derivedfrom Streptococcus pneumoniae (A), an expression vector cloning the same(B), and expression, isolation, and purification thereof (C).

FIG. 2 is a drawing comparing homology and similarity among amino acidsequences of a BgaC protein derived from Streptococcus pneumoniae, agalactosidase from Homo sapiens, and BgaC proteins from Bacillus cereusE33L, Carnobacterium piscicola BA, and Bacillus circulans.

FIG. 3 is a drawing showing the result of measuring galactosidase enzymeactivity of the BgaC protein, in which FIG. 3A is a graph showing enzymeactivity and quantitative values, in the case of using ONPG(o-nitrophenyl-D-galactopyranoside) or PNPG(p-nitrophenyl-D-galactopyranoside) as a substrate for measuring BgaCenzyme activity;

FIG. 3B is a graph showing an optimal pH range for enzyme activity; and

FIG. 3C is a graph showing an optimal temperature range for enzymeactivity.

FIG. 4 is a drawing showing the result of measuring the specificity andcleavage activity of the BgaC protein derived from Streptococcuspneumoniae for the sugar chain, in which the protein specificallyrecognizes and cleaves the sugar chain ofgalactose-beta1,3-N-acetylglucosamine (Galactose-β1,3-GlcNAc) (A),whereas the protein does not cleave the galactose linked toN-acetylgalactosamine (GalNAc) (B), and the non-reducing terminalgalactose linked by beta1,4 glycosidic linkage, instead of beta1,3glycosidic linkage (C).

FIG. 5 is a drawing showing the effect of the BgaC protein on the colonyformation of cancer cells, in which the colony formation is inhibitedwhen the cancer cells are treated with the BgaC protein, as comparedwith the cancer cells not treated with the BgaC protein, therebyoffering the possibility of using the BgaC protein for cancer cellgrowth inhibition.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to a novel beta-galactosidase derived fromStreptococcus pneumoniae R6, and to a method for using the enzyme inmodification and analysis of a sugar chain.

The present inventors used the genomic information of Streptococcuspneumoniae R6 strain, which is a pathogenic microorganism, in order tofind out a nucleic acid encoding a novel enzyme useful for sugar chainmodification. In order to find out a nucleic acid that encodes an enzymecapable of modifying the sugar chain derived from human, they screenedStreptococcus pneumoniae, which is a pathogenic microorganism causingpneumonia, and could easily use the genome of R6 strain that has alreadybeen disclosed.

There is a report that Streptococcus pneumoniae R6 has a bgaA geneexpressing a cell surface protein with beta1,4-galactosidase activity(Zahner and Hakenberck, J. Bacteriol. 182: 5919-5921, 2000). Anothergene, bagC with putative beta-galactosidase activity has been known toexist by comparing the sequence homology. However, the specific activityand biochemical characteristics of bgaC gene have not yet been reported.

Therefore, the present inventors amplified the gene designated as bgaCby polymerase chain reaction (PCR) using a chromosome of Streptococcuspneumoniae as a template, and cloned the gene, and isolated an nucleicacid molecule, so as to identify that the nucleic acid molecule hasbeta-galactosidase enzyme activity. The amino acid sequence of theidentified nucleic acid was represented by SEQ ID NO: 2, and its DNAsequence was represented by SEQ ID NO: 1.

Accordingly, in one embodiment, the present invention relates to annucleic acid molecule encoding the protein represented by SEQ ID NO: 2,or an nucleic acid molecule encoding a protein exhibitingbeta-galactosidase enzyme activity with at least 90% homology therewith.

The term “homology”, as used in beta-galactosidase gene derived fromStreptococcus pneumoniae of the present invention, refers to sequencesimilarity with DNA sequence of a wild type, and comprises a DNAsequence having preferably at least 90% homology with the DNA sequenceencoding the beta-galactosidase of the present invention. The homologycomparison was performed using comparison programs easily available. Thecommercially available computer programs can calculate the percentage ofthe homology (%) between two or more sequences, and homology (%) may becalculated over contiguous sequences.

In another embodiment, the present invention relates to an expressionvector containing the nucleic acid molecule of the invention. Theexpression vector is preferably pET28a-bgaC cloned into the E. coliexpression vector, pET28a (FIG. 1).

The term “vector” as used herein means any vehicle to allow DNAinsertion into a host cell, and includes all of the typical vectors suchas plasmid vector, cosmid vector, bacteriophage vector, and virusvector.

Further, the term “expression vector” as used herein means a vectorexpressing a target protein in a suitable host cell, and refers to agenetic construct including essential regulatory elements operablylinked to express a gene insert.

In still another embodiment, the present invention relates to a hostcell transformed with the expression vector. Transformation includes anymethod for introducing a nucleic acid molecule into an organism, a cell,a tissue, or an organ, and can be performed using the suitable standardtechnology selected according to the host cell, as disclosed in the art.Examples of the method include electroporation, protoplast fusion,calcium phosphate (CaPO₄) precipitation, calcium chloride (CaCl₂)precipitation, agitation with silicon carbide fiber,agrobacterium-mediated transformation, PEG, dextran sulfate,lipofectamine, or the like, but are not limited thereto.

The expression level and modification of the protein may vary dependingon the host cell transformed, thus very suitable host cell for thepurpose can be selected and used. The host cell is preferably aprokaryotic cell, and preferred prokaryotic host cells includeEscherichia coli, Bacillus subtilis, Streptomyces, Pseudomonas, Proteusmirabilis, and Staphylococcus, but are not limited thereto. Further, alower eukaryotic cell such as fungus (for example, Aspergillus) andyeast (for example, Pichia pastoris, Saccharomyces cerevisiae,Schizosaccharomyces, Neurospora crassa) can be used.

In a preferred example of the invention, the cloned expression vector,pET28a-bgaC was transformed into Escherichia coli BL21 (DE3) strain(Novagen) (Deposit No.: KCTC 10956BP), and deposited at KCTC (KoreanCollection for Type Cultures, Korea Institute of Bioscience andBiotechnology, 52, Ueun-dong, Yusung-gu, Daejeon-si, Korea) on Jun. 7,2006.

The transformed host cell was cultured in a suitable medium to express aprotein derived from Streptococcus pneumoniae, with beta-galactosidaseactivity, and the protein was isolated, purified to produce the proteinexhibiting beta-galactosidase activity. The present inventors designatedthe protein, which is expressed from the bgaC gene derived fromStreptococcus pneumoniae and has beta-galactosidase activity, as BgaC.

Accordingly, in still another embodiment, the present invention relatesto the BgaC protein derived from Streptococcus pneumoniae, or the aminoacid sequence represented by SEQ ID NO: 2, or the protein exhibitingbeta-galactosidase enzyme activity with at least 90% homology therewith.

More particularly, the beta-galactosidase of the protein was derivedfrom Streptococcus pneumoniae, and produced by recombinant DNAtechnology, in which the beta-galactosidase has a) maximum activity inthe range of 20 to 40° C., b) maximum activity in the pH range of 5.0 to8.0, c) a molecular weight of 50 to 100 kDa, d) more reactivity andhigher substrate specificity to PNPG than to ONPG, and has a function oflinkage-specifically cleaving the sugar chain.

The beta-galactosidase enzyme activity of the protein was analyzed usingONPG (o-nitrophenyl-D-galactopyranoside) and PNPG(p-nitrophenyl-D-galactopyranoside) as a substrate. As a result, it wasfound that the BgaC protein derived from Streptococcus pneumoniae hashigher activity on PNPG than ONPG (FIG. 3). Further, in order todetermine the optimal pH for the BgaC protein, its enzyme activity wasmeasured. Furthermore, in order to analyze the change in the enzymeactivity depending on temperature, its enzyme activity was measured overwide ranges of temperature. Consequently, it was found that the BgaCprotein has maximum activity at pH 6.5, and in the temperature range of30 to 35° C. (Example 4).

In still another embodiment, the present invention relates to thebeta-galactosidase that selectively hydrolyzesgalactose-beta1,3-N-acetylglucosamine, and the protein is preferablyBgaC.

In order to analyze the characteristic of BgaC protein recognizing asugar chain, a galactose cleavage test was performed using a variety ofcomplex sugar chains as the substrate. As a result, it was found thatthe BgaC protein selectively cleaves only the non-reducing terminalgalactose linked by beta1,3-glycosidic linkage. In particular, theprotein was found to be specific to the sugar followed by thebeta1,3-glycosidic linkage. More particularly, in the case where thesugar followed by the beta1,3-glycosidic linkage isN-acetylgalactosamine (GalNAc), the protein cannot cleave the terminalgalactose. In the case where the sugar followed by thebeta1,3-glycosidic linkage is N-acetylglucosamine (GlcNAc), the proteincan cleave the terminal galactose. From the experimental results, it canbe seen that the BgaC protein selectively cleaves the non-reducingterminal galactose in the structure ofgalactose-β1,3-N-acetylglucosamine.

In still another embodiment, the present invention relates to a methodfor linkage-specifically cleaving the galactose and modified sugar chainlinked to galactose-beta 1,3-N-acetylglucosamine using thebeta-galactosidase.

The method for linkage-specifically cleaving the galactose linked togalactose-beta 1,3-N-acetylglucosamine using the BgaC protein can beuseful for the structural analysis of the sugar chain. For the analysisof the component or sequence of the sugar chain, a HPLC or massspectrometry has been widely used. However, for the structural analysisof linkage specificity, a linkage-specific glycosidase is required. Inparticular, the glycosidase specific to the sugar followed by glycosidiclinkage is more useful for the structural analysis. Until now,beta-galactosidases specific to galactose beta1,3 linkage has been knownto exist. However, the BgaC protein of the present invention is thefirst beta1,3 galactosidase specific to the galactose followed byN-acetylglucosamine.

Preferably, the present invention provides a method for analyzing asugar chain, in which the galactose and modified sugar chain linked togalactose-beta 1,3-N-acetylglucosamine are treated with the proteinexhibiting beta1,3-galactosidase activity to cleave the sugar chain.

In still another embodiment, the present invention provides a method fortreating or diagnosing a disease, in which the specific sugar chainspresent in cancer are cleaved using the BgaC protein having galactosecleavage activity specific to galactose-beta1,3-N-acetylglucosamine.

In still another embodiment, the present invention provides apossibility of using the BgaC protein as an anti-cancer agent or a tumorinhibitor, based on the ability of inhibiting the colony formation ofcancer cells.

An sLe^(a) sugar chain has been known to be excessively expressed on thesurface of cancer cells. The sLe^(a) sugar chain has a core structure ofgalactose-beta1,3-N-acetylglucosamine (Gal-β1,3-GlcNAc). Therefore, ifthe BgaC protein of the invention is used, the galactose of sLe^(a) orLe^(a) sugar chain can be selectively cleaved, so as to prevent cancerprogression.

Hereinafter, the present invention will be described in more detail withreference to Examples. However, these Examples are for illustrativepurposes only, and the invention is not intended to be limited by theseExamples.

EXAMPLE 1 Amplification of bgaC Gene from Streptococcus pneumoniae

A Streptococcus pneumoniae chromosome was isolated from Streptococcuspneumoniae (ATCC BBA-255D) using a known phenol extraction (Ushiro etal., J Dent Res 70: 1422-1426, 1991). Polymerase chain reaction (PCR)was performed using the extracted chromosomal DNA as a template, and apair of primer, bgaC-N(cgctagCATGACACGATTTGAGATACGAG) andbgaC-C(ggaagcttTCATAAGTTTTCCCCCTTTATATG), at which NheI and HindIIIenzyme restriction sites were artificially inserted, so as to prepare aDNA fragment containing bgaC with a size of 1.8 kb (see FIG. 1A).

The base sequence of the prepared DNA fragment was translated. As aresult, it was found that the Streptococcus pneumoniae bgaC gene encodesan intracellular protein consisting of 595 amino acids. From the resultof translating into the amino acid sequence, it was found that theStreptococcus pneumoniae BgaC protein has 29.8%, 54.3%, 65.4%, and 46.7%homologies and 40.8%, 58.1%, 41.6%, and 54.2% similarities with Homosapiens galactosidase, the BgaC proteins of Bacillus cereus E33L,Carnobacterium piscicola BA, and Bacillus circulans, respectively (seeFIG. 2).

EXAMPLE 2 Large-scale Expression, Isolation, and Purification of BgaCProtein

A recombinant vector pET28a (Novagen) was cleaved with NheI and HindIIIrestriction enzymes, and the PCR product, which is treated with the sameenzymes and amplified in Example 1, was introduced into the vector (FIG.1B). The prepared recombinant vector was transformed into E. coli BL21(DE3) strain. The transformed E. coli was precultured in 5 ml of LBliquid medium (1% Bacto Tripton, 1% Sodium chloride, 0.5% Yeast extract)at 37° C. for 16 hours. The precultured medium was inoculated into freshLB medium at a dilution of 1:100, and cultured at 37° C. When theabsorbance of the medium was 0.4 to 0.6 at 600 nm, IPTG (isopropylthiogalactoside) was added thereto to be a final concentration of 1 mM.Thus, the BgaC protein expression was induced and the medium wascultured at 18° C. for 24 hours. The cultured E. coli cells werecentrifuged, recovered, and then disrupted by sonication. The disruptedcells were centrifuged, and the supernatant was taken to be purified byaffinity chromatography with nickel-nitrilotriacetic acid column, so asto obtain the 69 kDa BgaC protein (see FIG. 1C).

EXAMPLE 3 Galactosidase Activity Test for BgaC Protein

The basic activity of the BgaC protein was confirmed using ONPG(o-nitrophenyl-D-galactopyranoside) and PNPG(p-nitrophenyl-D-galactopyranoside) as a substrate. 2.2 mg of BgaCprotein was mixed with a reaction mixture [90 mM sodium phosphate(NaPO₄) (pH 6.5), 10 mM magnesium chloride (MgCl₂), 45 mMbeta-mercaptoethanol, 0.3 mM ONPG or PNPG] to be 300 μl, and reacted at30° C. for 30 minutes. Then, its absorbance was measured at 420 nm todetermine the amount of ONP (o-nitrophenol) or PNP (p-nitrophenol)produced. The activity of BgaC protein (Unit) was defined as ability toconvert 1 nmole of ONPG or PNPG into ONP or PNP at 30° C. for 1 minute.The maximum reaction rate of the enzyme is 2.6 times higher in the caseof using PNPG than in the case of using ONPG as the substrate. Further,the substrate affinity of the enzyme is 3.5 times stronger for PNPG thanfor ONPG (see FIG. 3A). In accordance with the Example, it was foundthat the BgaC protein has more reactivity and higher substratespecificity to PNPG than to ONPG.

EXAMPLE 4 Determination of Optimal pH and Temperature for BgaC ProteinActivity

In order to determine an optimal range of hydrogen ion concentration(pH) for maximum enzyme activity, the enzyme activity of the BgaCprotein was measured at various ranges of pH, that is, at the pH rangeof 3.89 to 8. A sodium acetate buffer was used to adjust pH 6 or less,and a sodium phosphate buffer was used to adjust pH 6 or more.Beta-galactosidase activity was measured using ONPG or PNPG as thesubstrate only with changing pH of the buffer solution (3.89, 4.28, 4.6,5, 5.41, 6, 6.5, 7, 7.5, and 8) among reactants. The BgaC enzyme wasfound to have maximum activity at pH 6.5 (see FIG. 3B). In order tomeasure the change in enzyme activity of the BgaC protein according totemperature, the enzyme activity of the BgaC protein was measured invarious ranges of temperature. As a result, the BgaC enzyme was found tohave maximum activity in the temperature range of 30 to 35° C. (see FIG.3C).

EXAMPLE 5 Analysis of Sugar Chain Specificity and Activity of BgaCProtein

Substrate specificity of the BgaC protein was analyzed using sugarchains 042, 028 purchased from Takara Co. (Japan) and NA2 sugar chain[Mannotriose-di-(N-acetyl-D-glucosamine)]purchased from Sigma. The sugarchains from Takara Co. are sugar chains labeled with fluorescence, andthus a HPLC from Waters Co. (USA), which is equipped with fluorescencedetector, was used. The sugar chain from Sigma is a sugar chain notlabeled with fluorescence, and thus a microflex from Bruker Co.(Germany), which is a MALDI-TOF mass spectrometry, was used to analyze amass change. FIG. 4A is the result of HPLC analysis by treating thesugar chain 042 (Gal-β1,3-GlcNAc-β1,3-Gal-β1,4-Glc-PA) from Takara withthe BgaC protein. The BgaC protein hydrolyzed the non-reducing terminalgalactose of the sugar, so as to give a peak ofGlcNAc-β1,3-Gal-β1,4-Glc-PA as a product, in which the galactose wasremoved. FIG. 4B is the result of treating the sugar chain 028(Gal-β1,3-GalNAc-β1,3-Gal-β1,4-Glc-PA) from Takara with the BgaCprotein, in which the terminal galactose of the sugar was found not tobe removed. FIG. 4C is the result of MALDI-TOF analysis by treating thesugar chain NA2 from sigma with the BgaC protein. The sugar chain NA2has a bi-antennary structure with two branches, in which thenon-reducing terminal portion is Gal-β1,4-GlcNAc-β1,3-Man. It was foundthat the BgaC protein cannot cleave the terminal galactose linked bybeta1,4 glycosidic linkage.

EXAMPLE 6 Ability of BgaC Protein to Inhibit Colony Formation of CancerCells

In order to induce the overexpression of RAS protein, which is involvedin tumor development, a fibroma cell line, NIH3T3 transfected with rasgene was inoculated into a 10 mm culture plate, in which Dulbecco'smodified eagle's medium (DMEM) containing 20% fetal bovine serum and 300unit of the BgaC protein was put, and cultured at 37° C. with 5% CO₂.The overexpression of ras gene promotes the colony formation of NIH3T3cells. The colony formation of NIH3T3 cells, which had been treated withthe BgaC protein, was inhibited, as compared with that of NIH3T3 cellsthat had not been treated with the BgaC protein (see FIG. 5). The colonyformation is a general characteristic of cancer cells. Sialyl Lewis A(sLe^(a)), which is a Lewis antigen acting as a ligand involved in themetastasis of cancer cells, is a sugar chain formed bygalactose-beta1,3-N-acetylglucosamine(Gal-β1,3-GlcNAc) linkage (Takadaet al., Cancer Res. 53: 354-361, 1993). Accordingly, the experimentalresult indicates that the BgaC protein blocked the formation of Lewis Aantigen, so as to reduce the metastasis of caner cells, and thus thecolony formation was inhibited. The colony formation of cancer cells wasinhibited by the BgaC activity, which offers a possibility of using theBgaC protein as an anti-cancer agent or a tumor inhibitor.

EFFECTS OF THE INVENTION

As described above, the beta-galactosidase, BgaC protein canlinkage-specifically cleave a sugar chain, complex sugar, andoligosaccharide containing galactose-beta1,3-N-acetylglucosamine,thereby being useful for linkage-specific analysis of the sugar chains.Further, the specific sugar chains are selectively cleaved using theBgaC protein having the galactose cleavage activity specific to thesugar chains, which offers a possibility of using the protein medicallyor diagnostically such as an anti-cancer agent or a tumor inhibitor.Thus, the present invention is very useful for the pharmaceuticalindustry.

1. A protein exhibiting beta-galactosidase enzyme activity, which has anamino acid sequence represented by SEQ ID NO: 2, or an amino acidsequence having at least 90% homology therewith.
 2. The proteinaccording to claim 1, wherein the beta-galactosidase selectivelyhydrolyzes the galactose in the structure of galactose-β1,3-N-acetylglucosamine.
 3. The protein according to claim 1, which hasthe following characteristics of (a) to (d): (a) maximum activity in thetemperature range of 20 to 40° C.; (b) maximum activity at a pH range of5.0 to 8.0; (c) a molecular weight of 50 to 100 kDa; and (d) morereactivity and higher substrate specificity to PNPG than to ONPG.
 4. Anisolated nucleic acid molecule encoding the protein of claim
 1. 5. Anexpression vector comprising the nucleic acid molecule of claim
 4. 6.The expression vector according to claim 5, shown in FIG. 1B.
 7. Atransformant transformed by the expression vector of claim
 6. 8. Thetransformant according to claim 7, which has a deposit number ofKCTC10956BP.
 9. A method for linkage-specifically cleaving the galactoseand modified sugar chain in the structure of galactose-beta1,3-N-acetylglucosamine using the protein according to any one of claims1 to
 3. 10. A method for linkage-specifically analyzing the structure ofa sugar chain by treating with the protein according to any one ofclaims 1 to
 3. 11. A composition for inhibiting tumor, comprising theprotein according to any one of claims 1 to 3 as an active ingredient.